Reaction tank for nucleic acid amplification, cartridge, device, and nucleic acid amplification treatment method

Abstract

The present invention is a sealed nucleic acid amplification reaction tank 20 provided with a reaction solution supply port 23 and a gas discharge unit 24. In the reaction tank 20, the lower space is a reaction solution storage space 21, and the upper space is a reaction solution supply space 22. In the reaction tank according to one aspect of the present invention, the ratio (volume ratio V2 / V1) of the volume (V2) of the reaction solution supply space to the volume (V1) of the reaction solution storage space is 3.0 or less. In the reaction tank according to another aspect of the present invention, the gas discharge unit 24 has a ventilation passage 24b extending from the gas discharge port 24a downstream of the gas discharge port 24a. The present invention provides: a reaction tank and a cartridge which can be used for testing using nucleic acid amplification; a nucleic acid amplification device equipped with the reaction tank and the cartridge; and a nucleic acid amplification treatment method using them.

Description

Reaction chamber, cartridge, device for nucleic acid amplification, and nucleic acid amplification treatment method

[0001] The present invention relates to a reaction vessel, cartridge, device, and nucleic acid amplification processing method for nucleic acid amplification. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2023-123721, filed on July 28, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] With the spread of COVID-19, there is a growing demand for technology that can easily and reliably determine whether or not a person is infected with a virus by collecting, amplifying, and detecting nucleic acids from the virus. Even after the COVID-19 problem is resolved, humanity will continue to face various viral and bacterial diseases, and the demand for similar technology will continue to exist.

[0003] With the advent of PCR using a thermocycler and the subsequent development of various methods other than thermocycle PCR, nucleic acid amplification and detection techniques have become largely established as methodologies, and are now easily performed not only in laboratories but also in hospital testing labs. However, even so, nucleic acid extraction from biological samples such as viruses is still often performed manually, followed by nucleic acid amplification and detection using existing amplification and detection devices. However, with the spread of COVID-19, it is clear that the seriousness of this situation cannot be addressed by using existing methods and devices and simply bringing samples to public health centers or testing companies.

[0004] The present applicant has filed patent applications for a nucleic acid amplification cartridge that can process a large amount of specimens quickly and easily, regardless of location, to determine the presence or absence of viral infection (Patent Documents 1 to 4).

[0005] Patent Document 1 discloses a cartridge 60 containing multiple nucleic acid amplification reaction chambers 50 and a nucleic acid amplification method using the same. As shown in FIG. 5 , the reaction chambers 50 are elongated, with a reaction solution storage space 51 at the bottom and a reaction solution supply space 52 at the top. The reaction solution supply space 52 has a reaction solution supply port 53 and an exhaust port 54, with the reaction solution supply port 53 located longitudinally below the exhaust port 54 (claim 1). The cartridge 60 in Patent Document 1 contains multiple (four) reaction chambers 50. The reaction solution supply port 53 of each reaction chamber is connected to a flow path 55 that transports the reaction solution used in the nucleic acid amplification reaction. The flow path 55 divides the reaction solution at a branch point and supplies the reaction solution approximately evenly from the reaction solution supply ports of each reaction chamber. The exhaust port 54 is connected to an external exhaust mechanism via an exhaust space on the back side of the cartridge.

[0006] The cartridge 60 is used while attached to a nucleic acid amplification device having an exhaust mechanism and a heating mechanism. When the exhaust mechanism exhausts air, the inside of the reaction chamber 50 is controlled to a negative pressure via the exhaust port 54. When the inside of the reaction chamber 50 is controlled to a negative pressure, the reaction solution containing the extract from the specimen is drawn to the negative pressure side, moves through the flow path 55, and is supplied from the reaction solution supply port 53 to the reaction solution supply space 52, and is then contained in the reaction solution containing space 51. Then, with the reaction solution contained in the reaction solution containing space 51, the heating mechanism begins to heat the reaction solution containing space 51, and the reaction solution is subjected to a nucleic acid amplification reaction.

[0007] Patent Document 1: JP 2023-026235 A Patent Document 2: JP 2023-026236 A Patent Document 3: JP 2023-026237 A Patent Document 4: JP 2023-026238 A The entire disclosures of Patent Documents 1 to 4 are incorporated herein by reference.

[0008] By using the cartridge described in Patent Document 1, it has become possible to process a large amount of specimens quickly and easily, regardless of location, and determine the presence or absence of viral infection. However, it has been found that when a viral test is performed using the cartridge described in Patent Document 1, test abnormalities such as false negatives or false positives may occur.

[0009] The present invention has been made in view of the above-mentioned problems, and has as its object to provide a reaction vessel and cartridge that can suppress the occurrence of test abnormalities in tests that utilize nucleic acid amplification.

[0010] Another object of the present invention is to provide a nucleic acid amplification device and a nucleic acid amplification method that can suppress the occurrence of test abnormalities in tests that utilize nucleic acid amplification by using the above-mentioned reaction vessel and cartridge.

[0011] The above problem was solved by reducing the proportion of the reaction solution supply space above the reaction vessel compared to conventional methods. The above problem was also solved by providing an air vent downstream of the exhaust port of the reaction vessel, extending from the exhaust port. The present invention is as follows: <1> A sealed nucleic acid amplification reaction vessel equipped with a reaction solution supply port and an exhaust section, wherein the exhaust section has an exhaust port, the lower space of the reaction vessel is a reaction solution storage space, the upper space is a reaction solution supply space, the reaction solution supply port and the exhaust port are located on the inner wall surface of the reaction solution supply space, and the reaction solution supply port is located below the exhaust port, and the ratio of the volume (V2) of the reaction solution supply space to the volume (V1) of the reaction solution storage space (volume ratio V2 / V1) is 3.0 or less. <2> The reaction vessel according to <1>, wherein the volume ratio V2 / V1 is 2.5 or less. <3> The reaction vessel according to <1> or <2>, wherein at least a portion of the upper surface of the inner wall of the reaction vessel is inclined. <4> The reaction vessel according to any one of <1> to <3>, wherein at least the inner wall surface of the reaction solution supply space is water-repellent. <5> The reaction vessel according to any one of <1> to <4>, wherein the exhaust unit further has an air vent extending from the exhaust port downstream of the exhaust port. <6> A sealed reaction vessel for nucleic acid amplification equipped with a reaction solution supply port and an exhaust unit, wherein the exhaust unit has an exhaust port and an air vent extending from the exhaust port downstream of the exhaust port, the lower space of the reaction vessel is a space for containing reaction solution, the upper space is a space for supplying reaction solution, the reaction solution supply port and the exhaust port are located on the inner wall surface of the reaction solution supply space, and the reaction solution supply port is located below the exhaust port. <7> The reaction vessel according to <5> or <6>, wherein the ratio of the length to the width of the air vent channel is 1 to 40. <8> The reaction vessel according to any one of <5> to <7>, wherein the length of the air vent channel is 2 to 8 mm. <9> The reaction vessel according to any one of <5> to <8>, wherein the width of the vent channel is 0.2 to 2.0 mm. <10> The reaction vessel according to any one of <5> to <9>, wherein the inner wall surface of the vent channel is water-repellent. <11> The reaction vessel according to any one of <1> to <10>, wherein the exhaust port is located above the inner wall of the reaction vessel.<12> A nucleic acid amplification cartridge for subjecting a reaction solution to a nucleic acid amplification reaction, comprising at least one reaction chamber for accommodating a reaction solution and a flow path for supplying the reaction solution to the reaction chamber, wherein the reaction chamber is the reaction chamber described in any one of <1> to <11>, and the flow path is connected to a reaction solution supply port of the reaction chamber. <13> The cartridge described in <12>, wherein the number of reaction chambers is two or more, the flow path is divided at a branch point and connected to the reaction solution supply port of each reaction chamber, and the cartridge further comprises an exhaust gas junction space having a junction point where all exhaust gas flows from each reaction chamber are joined into one flow, and the volumes of the spaces through which each exhaust gas passes from the exhaust port of each reaction chamber to the junction point are approximately equal to each other. <14> The cartridge according to <12> or <13>, wherein the number of reaction vessels is two or more, the flow path is divided at a branch point and connected to the reaction solution supply port of each reaction vessel, and for each reaction vessel, the length of the flow path from the branch point closest to the reaction vessel to the reaction solution supply port is longer than the linear distance from the branch point to the reaction solution supply port. <15> The cartridge according to any one of <12> to <14>, wherein the number of reaction vessels is two or more, the flow path is divided at a branch point and connected to the reaction solution supply port of each reaction vessel, and for each reaction vessel, the position of the branch point closest to the reaction vessel is the same as the position of the exhaust unit or higher than the position of the exhaust unit. <16> A nucleic acid amplification device comprising: an attachment unit for attaching the cartridge according to <12>, an exhaust mechanism for evacuating the inside of the reaction vessels of the cartridge, and a heating mechanism for heating the reaction solution storage space and the reaction solution supply space of the cartridge. <17> A nucleic acid amplification treatment method using the cartridge described in <12>, comprising: supplying a reaction solution from the reaction solution supply port to a reaction solution supply space by creating a negative pressure inside the reaction tank by suction from an exhaust port; and subjecting the reaction solution that spontaneously moves from the reaction solution supply space to the reaction solution storage space to a nucleic acid amplification reaction.

[0012] According to the present invention, the occurrence of test abnormalities in tests that utilize nucleic acid amplification can be suppressed.

[0013] Fig. 1 is an enlarged view of the front side of a portion of a cartridge having a nucleic acid amplification reaction chamber according to one embodiment, the portion being used for nucleic acid amplification reaction. Fig. 2 is an enlarged view of the back side of the portion of the cartridge shown in Fig. 1, the portion being used for nucleic acid amplification treatment. Figs. 3AB and 3CD (a) to (d) show test results when a cartridge according to one embodiment of the present invention was used. Fig. 4 (a) to (c) show test results when a conventional cartridge was used. Fig. 5 is an enlarged view of the portion of a conventional nucleic acid amplification cartridge, the portion being used for nucleic acid amplification reaction.

[0014] <Nucleic Acid Amplification Reaction Chamber> The present invention broadly includes first to third embodiments of nucleic acid amplification reaction chambers.

[0015] A first aspect of the nucleic acid amplification reaction vessel of the present invention is as follows: A sealed nucleic acid amplification reaction vessel equipped with a reaction solution supply port and an exhaust part, wherein the exhaust part is an exhaust port, the lower space of the reaction vessel is a reaction solution storage space, the upper space is a reaction solution supply space, the reaction solution supply port and the exhaust port are located on the inner wall surface of the reaction solution supply space, and the reaction solution supply port is located below the exhaust port, and the ratio of the volume (V2) of the reaction solution supply space to the volume (V1) of the reaction solution storage space (volume ratio V2 / V1) is 3.0 or less.

[0016] A second aspect of the nucleic acid amplification reaction vessel of the present invention is as follows: A sealed nucleic acid amplification reaction vessel equipped with a reaction solution supply port and an exhaust section, wherein the exhaust section has an exhaust port and an air passage extending from the exhaust port downstream of the exhaust port, the lower space of the reaction vessel is a space for containing reaction solution, the upper space is a space for supplying reaction solution, the reaction solution supply port and the exhaust port are located on the inner wall surface of the space for supplying reaction solution, and the reaction solution supply port is located below the exhaust port.

[0017] A nucleic acid amplification reaction vessel according to a third aspect of the present invention is a reaction vessel that combines the feature of the first aspect (a volume ratio V2 / V1 of 3.0 or less) and the feature of the second aspect (an air vent extending from an exhaust port), and is as follows: A sealed nucleic acid amplification reaction vessel equipped with a reaction solution supply port and an exhaust section, wherein the exhaust section has an exhaust port and an air vent extending from the exhaust port downstream of the exhaust port, the lower space of the reaction vessel is a space for containing reaction solution, the upper space is a space for supplying reaction solution, the reaction solution supply port and the exhaust port are located on the inner wall surface of the space for supplying reaction solution, and the reaction solution supply port is located below the exhaust port, and the ratio (volume ratio V2 / V1) of the volume of the space for containing reaction solution (V1) to the volume of the space for supplying reaction solution (V2) is 3.0 or less.

[0018] The nucleic acid amplification reaction chamber of the present invention will be described in detail below based on one embodiment of the nucleic acid amplification reaction chamber 20 shown in Figures 1 and 2. Figure 1 shows four reaction chambers 20 as an example.

[0019] <<Reaction Tank of First Aspect>> A reaction tank of the first aspect will be described. The reaction tank 20 is elongated and sealed, with the lower space being a reaction liquid storage space 21 and the remaining upper space being a reaction liquid supply space 22 having an exhaust port 24. In the first aspect, the exhaust port 24 is an exhaust port 24a, and the ratio of the volume (V2) of the reaction liquid supply space to the volume (V1) of the reaction liquid storage space (volume ratio V2 / V1) is 3.0 or less. The reaction liquid supply space 22 has a reaction liquid supply port 23 and an exhaust port 24a on its inner wall surface, with the reaction liquid supply port 23 located longitudinally below the exhaust port 24a. For example, the reaction liquid supply port 23 can be located on a side wall within the reaction liquid supply space 22, and the exhaust port 24a can be located on another side wall within the reaction liquid supply space 22. The cartridge can have an exhaust space 41 on the back side that is in communication with an external exhaust mechanism, and in a first embodiment, the exhaust port 24a can be in direct communication (through the front and back) with the exhaust space 41. The reaction solution supply port 23 is in communication with a flow path 30 that transports the reaction solution.

[0020] In this specification, "below" refers to the side of the reaction vessel that faces the reaction liquid receiving space in the longitudinal direction, whereas "upper" refers to the side of the reaction vessel that faces the reaction liquid supply space in the longitudinal direction. In this specification, the terms "downstream" and "upstream" may be used to describe the positional relationship of components in the reaction vessel and cartridge. "Downstream" means downstream in the direction along the flow of gas in the reaction vessel 20 discharged through the exhaust section 24 and the flow of liquid in the flow path resulting from this flow, and "upstream" means upstream in the direction along these flows.

[0021] When exhaust is performed through the exhaust space 41 and the exhaust port 24a, a negative pressure is applied to the reaction vessel 20 and further to the flow channel 30. The reaction solution is drawn to the negative pressure side and flows through the flow channel 30 while being divided at branch points 31a and 31b of the flow channel 30. The divided reaction solution is supplied to the reaction solution supply space 22 from the reaction solution supply port 23 of each reaction vessel 20, and then contained in the reaction solution containing space 21. Then, with the reaction solution contained in the reaction solution containing space 21, heating of the reaction solution containing space 21 (or both the reaction solution containing space 21 and the reaction solution supply space 22) begins, and the reaction solution is subjected to a nucleic acid amplification reaction.

[0022] The reaction solution is a sample solution that contains or may contain nucleic acid to be amplified, and for example, when testing a specimen, the reaction solution contains an extract from the specimen and may further contain reagents such as an enzyme, primer or probe corresponding to the nucleic acid to be amplified, and a buffer. If the reaction solution contains nucleic acid to be amplified, the nucleic acid will be amplified by the nucleic acid amplification reaction.

[0023] The reaction vessel can be sealed, for example, by producing a cartridge body by resin molding, the cartridge body having recesses on its surface that define spaces such as the reaction liquid storage space 21, the reaction liquid supply space 22, and even the flow path, and then attaching a film to the surface of the cartridge body.

[0024] The reaction liquid containing space 21 is a space for containing a reaction liquid and amplifying the nucleic acid contained in the reaction liquid. The volume of the reaction liquid containing space is set to be approximately equal to the amount of reaction liquid contained in the reaction liquid containing space. The volume of the reaction liquid containing space is, for example, in the range of 10 to 500 μL, preferably in the range of 10 to 100 μL, taking into consideration that the amount of sample that can be used in the test is very small, and that the very small amount of nucleic acid in the reaction liquid needs to be amplified and appropriately detected.

[0025] The reaction solution supply space 22 is a space for taking in the reaction solution from the reaction solution supply port 23 and then supplying the reaction solution to the reaction solution storage space 21. The volume of the reaction solution supply space is set so that the volume ratio V2 / V1 is 3.0 or less, from the viewpoint of suppressing the occurrence of test abnormalities in tests using nucleic acid amplification. From the viewpoint of further suppressing the occurrence of test abnormalities, the volume ratio V2 / V1 is preferably 2.5 or less, 2.0 or less, or 1.8 or less. There is no particular restriction on the lower limit of the volume ratio V2 / V1, but it is practical to set it to 1.0 or more, and it may also be 1.1 or more, or 1.2 or more.

[0026] In the nucleic acid amplification reaction vessel of the first aspect, the volume ratio V2 / V1 is 3.0 or less, so that the occurrence of test abnormalities in tests utilizing nucleic acid amplification can be suppressed.

[0027] Although the exact cause of the test abnormality has not been identified, the inventors' investigations have revealed that when the reaction solution is subjected to a nucleic acid amplification reaction, some of the water may leak out of the reaction solution, reducing the volume of the reaction solution in the reaction solution storage space. This reduction in the reaction solution volume may have changed the reaction solution concentration, causing the test abnormality.

[0028] While not intending to be bound by theory, the following reasons are considered to be the cause of water leakage from the reaction solution. The downstream side of the exhaust port 24a in the reaction solution supply space 22 is closed during the nucleic acid amplification reaction, preventing the flow of gas due to exhaust from the external exhaust mechanism. However, it was confirmed that water vapor from the reaction solution evaporates and remains in the reaction solution supply space above the reaction chamber as water vapor, and that the water vapor condenses on the inner wall surface of the reaction solution supply space and remains as water droplets. In addition, in the conventional cartridge, water droplets were also observed on the inner wall surface of the exhaust space downstream of the reaction chamber. This is thought to be because the conventional cartridge makes it easier for water vapor to leak downstream of the reaction chamber. Based on these facts, possible causes of water leakage from the reaction solution are presumed to be, for example, water vapor retention in the reaction solution supply space, water droplets remaining on the inner wall surface of the reaction solution supply space, and water vapor leaking downstream from the reaction solution supply space.

[0029] In the nucleic acid amplification reaction vessel of the first embodiment, the volume ratio V2 / V1 is 3.0 or less, and the volume of the reaction solution supply space is set smaller than that of the reaction vessel of the cartridge described in Patent Document 1. In the cartridge of Patent Document 1 (Figure 5 in the present drawings), the volume ratio V2 / V1 is approximately 5.4. In the reaction vessel of the first embodiment, the volume ratio V2 / V1 is 3.0 or less, thereby reducing the volume and inner wall surface area of ​​the reaction solution supply space, thereby reducing the amount of water vapor remaining in the reaction solution supply space and the amount of water droplets remaining on the inner wall surface of the reaction solution supply space. As a result, it is believed that water leakage from the reaction solution during the nucleic acid amplification reaction is suppressed, thereby suppressing the occurrence of test abnormalities in tests using nucleic acid amplification.

[0030] The reaction solution supply space 22 has an exhaust section 24 for exhausting the air from inside the reaction vessel, and in the first embodiment, the exhaust section 24 is an exhaust port 24a. The exhaust port 24a can be directly connected to the exhaust space 41 on the back side. The exhaust port 24a is preferably located in the upper longitudinal direction of the reaction vessel (or reaction solution supply space) (e.g., above the middle of the reaction solution supply space in the longitudinal direction), and more preferably on the upper surface of the inner wall of the reaction solution supply space. By ensuring a distance between the exhaust port 24a and the reaction solution storage space, it is possible to prevent the reaction solution supplied from the reaction solution supply port 23 from bouncing back and flowing into the exhaust port 24a.

[0031] From the viewpoint of further suppressing the occurrence of test abnormalities, it is preferable that at least a portion of the upper surface of the inner wall of the reaction vessel (or reaction liquid supply space) is inclined. By inclining at least a portion of the upper surface of the inner wall, water droplets condensed on the upper surface of the inner wall can easily flow along the inner wall surface, facilitating the recovery of water into the reaction liquid. A preferred angle of inclination is, for example, 105 to 145 degrees, more preferably 115 to 135 degrees. In addition, from the viewpoint of further suppressing the occurrence of test abnormalities, it is preferable that at least the inner wall surface of the reaction liquid supply space is treated to be water-repellent, and the entire inner wall of the reaction vessel may be treated to be water-repellent. The water-repellent treatment makes it easier for water droplets condensed on the inner wall surface of the reaction liquid supply space to flow along the inner wall surface, facilitating the recovery of water into the reaction liquid.

[0032] The reaction vessel 20 may have a protrusion 25 that protrudes toward the inside of the reaction vessel so as to reduce the diameter at the boundary between the reaction liquid accommodating space 21 and the reaction liquid supply space 22. The presence of the protrusion 25 is expected to have the effect of suppressing the generation of bubbles in the reaction liquid when the reaction liquid is supplied from the reaction liquid supply port 23. Furthermore, the opening 26 formed by the protrusion 25 serves as an entrance through which the reaction liquid supplied to the reaction liquid supply space 22 moves to the reaction liquid accommodating space 21, and also has the function of defining the area where the liquid surface of the reaction liquid after moving to the reaction liquid accommodating space 21 comes into contact with the air in the reaction liquid supply space 22. By setting the volume of the reaction liquid so that the liquid surface of the reaction liquid is positioned at approximately the same height as the opening 26, the liquid surface of the reaction liquid becomes approximately equal to the area of ​​the opening 26 and is smaller than when the protrusion 25 is not present, thereby making it easier for the reaction liquid to remain in the reaction liquid accommodating space 21.

[0033] When the horizontal cross-sectional area of ​​the reaction vessel (for example, the cross-sectional area at the position where the reaction liquid supply port 23 is located) is taken as 100, the ratio of the horizontal cross section at the position where the protrusion 25 is located, i.e., the area of ​​the opening 26, is preferably in the range of 30 to 95, more preferably in the range of 60 to 80. The area of ​​the opening 26 in the horizontal cross section at the position where the protrusion 25 is located varies depending on the horizontal cross-sectional area of ​​the reaction vessel, but is, for example, 1 to 10 mm ２ The horizontal cross-sectional area of ​​the reaction vessel can be, for example, in the range of 2 to 20 mm ２ However, it is not intended to be limited to these numerical ranges.

[0034] From the viewpoint of detecting the amplified nucleic acid directly after or during nucleic acid amplification, it is preferable that at least a portion of the side wall of the reaction solution containing space be optically transparent. For example, a film attached to the surface of the cartridge body can be optically transparent. Furthermore, the horizontal cross-sectional area of ​​the reaction solution containing space (e.g., the largest horizontal cross-sectional area within the reaction solution containing space) can be smaller than the horizontal cross-sectional area of ​​the reaction solution supply space (e.g., the largest horizontal cross-sectional area within the reaction solution supply space). In particular, when at least a portion of the side wall is optically transparent, it is preferable to shorten the distance to the inner surface of the wall facing the optically transparent side wall so that the horizontal cross-sectional area of ​​the reaction solution containing space is smaller than the horizontal cross-sectional area of ​​the reaction solution supply space. This allows for a reduction in the amount of reaction solution to be contained in the reaction solution containing space, and allows for efficient use of detection light (excitation light) for the reaction solution containing the amplified nucleic acid.

[0035] <<Reaction Tank of Second Aspect>> The reaction tank of the second aspect will be described. The reaction tank of the second aspect differs from the reaction tank of the first aspect in that the volume ratio V2 / V1 is not required to be 3.0 or less, and the reaction tank of the second aspect has an air passage 24b extending from the exhaust port 24a downstream of the exhaust port 24a. The other configurations are the same as those of the reaction tank of the first aspect unless otherwise specified.

[0036] The reaction vessel 20 is elongated and sealed, with the lower space being a reaction liquid storage space 21 and the remaining upper space being a reaction liquid supply space 22 having an exhaust section 24. In a second embodiment, the exhaust section 24 has an exhaust port 24a and an air vent 24b extending from the exhaust port downstream of the exhaust port. The reaction liquid supply space 22 has a reaction liquid supply port 23 and an exhaust port 24a on its inner wall surface, with the reaction liquid supply port 23 located longitudinally below the exhaust port 24a. The air vent 24b is connected to an air vent 24c for venting the inside of the reaction vessel 20. As shown in FIG. 2 , the cartridge has an exhaust space 41 on the back side that is connected to an external exhaust mechanism, and the air vent 24c can be directly connected to the exhaust space 41.

[0037] When exhaust is performed through the exhaust space 41, the vent 24c, the ventilation path 24b, and the exhaust port 24a, similarly to the first embodiment, a negative pressure is applied to the reaction vessel 20 and further to the flow path 30, and the reaction liquid is accommodated in each reaction liquid accommodation space 21. Then, with the reaction liquid accommodated in the reaction liquid accommodation space 21, heating of the reaction liquid accommodation space 21 (or both the reaction liquid accommodation space 21 and the reaction liquid supply space 22) begins, and the reaction liquid is subjected to a nucleic acid amplification reaction.

[0038] The volume of the reaction solution supply space is not particularly limited and is, for example, in the range of 10 to 500 μL, preferably in the range of 10 to 100 μL. In the reaction vessel of the second embodiment, from the viewpoint of suppressing the occurrence of test abnormalities in tests using nucleic acid amplification, the exhaust section 24 of the reaction solution supply space 22 has an exhaust port 24a and an air vent 24b extending from the exhaust port downstream of the exhaust port. The cross-sectional shape perpendicular to the length of the air vent is not particularly limited and may be rectangular, circular, semicircular, triangular, or other polygonal shapes. From the viewpoint of further suppressing the occurrence of test abnormalities, the length of the air vent is preferably 2 to 8 mm, more preferably 3 to 6 mm, and the width of the air vent (the length perpendicular to the longitudinal direction of the reaction vessel and parallel to the cartridge surface; for example, the length of the air vent in the left-right direction in FIG. 1 ) is preferably 0.2 to 2.0 mm, more preferably 0.3 to 1.5 mm. From the same viewpoint, the ratio of the length to the width of the ventilation channel is preferably 1 to 40, more preferably 3 to 10. The height of the ventilation channel (the length in the direction perpendicular to the length and width directions of the ventilation channel; for example, the length of the ventilation channel in the direction perpendicular to the paper surface in FIG. 1) is preferably 0.2 to 2.0 mm, more preferably 0.3 to 1.5 mm. The cross-sectional area of ​​the ventilation channel in the cross section perpendicular to the length direction is 0.1 to 2.0 mm. ２ It is preferable that the thickness is 0.2 to 1.0 mm. ２ The vent hole 24c is, for example, a circular hole, and the diameter of the hole can be slightly wider than the width of the air passage. The diameter of the vent hole 24c is, for example, 1 to 3 mm, and may be 1.2 to 2 mm.

[0039] The nucleic acid amplification reaction vessel of the second embodiment has an air vent extending from the exhaust port downstream of the exhaust port, thereby suppressing the occurrence of test abnormalities in tests using nucleic acid amplification. As described above, the test abnormalities may have occurred due to a portion of the water leaking out of the reaction solution, reducing the volume of the reaction solution. However, water vapor tends to accumulate in the air vent, and once the air vent is filled with water vapor, further inflow of water vapor into the air vent is thought to be inhibited. Thus, in the second embodiment, the presence of the air vent reduces the amount of water vapor leaking downstream from the reaction solution supply space during the nucleic acid amplification reaction, which is thought to have suppressed the occurrence of test abnormalities in tests using nucleic acid amplification.

[0040] The ventilation path 24b preferably extends upward along the longitudinal direction of the reaction vessel, which allows water droplets condensed on the inner wall surface of the ventilation path to easily move down the inner wall surface due to their own weight, facilitating the recovery of water into the reaction solution. In this case, from the viewpoint of further suppressing the occurrence of test abnormalities, the length of the ventilation path is preferably 0.1H to 1.0H, and more preferably 0.4H to 0.8H, where H is the height of the reaction vessel in the longitudinal direction.

[0041] The inner wall surface of the ventilation channel is preferably treated with a water-repellent finish, which allows water droplets condensed on the inner wall surface of the ventilation channel to easily flow along the inner wall surface, facilitating recovery of moisture into the reaction solution.In addition, the length, width, height, and cross-sectional shape of the ventilation channel of each reaction vessel are preferably the same so that negative pressure is applied uniformly to each reaction vessel.

[0042] <<Reaction Tank of Third Aspect>> The reaction tank of the third aspect will be described. The reaction tank of the third aspect differs from the reaction tank of the first aspect in that the volume ratio V2 / V1 is 3.0 or less and that the reaction tank of the third aspect has an air vent 24b extending from the exhaust port 24a downstream of the exhaust port 24a. Unless otherwise specified, the other configuration is the same as that of the reaction tank of the first aspect, and the details of the air vent are the same as those of the reaction tank of the second aspect.

[0043] The reaction vessel 20 is elongated and sealed, with the lower longitudinal space being the reaction liquid storage space 21 and the remaining upper space being the reaction liquid supply space 22 having an exhaust section 24. In a third embodiment, the exhaust section 24 has an exhaust port 24a and an air vent 24b extending from the exhaust port downstream of the exhaust port. The ratio of the volume (V2) of the reaction liquid supply space to the volume (V1) of the reaction liquid storage space (V1) (volume ratio V2 / V1) is 3.0 or less. The volume of the air vent 24b is not included in V2. The reaction liquid supply space 22 has a reaction liquid supply port 23 and an exhaust port 24a on its inner wall surface, with the reaction liquid supply port 23 located longitudinally below the exhaust port 24a. The air vent 24b is connected to an air vent 24c for exhausting the air from the reaction vessel 20. As shown in FIG. 2, the cartridge has an exhaust space 41 on the rear side thereof, which is connected to an external exhaust mechanism, and the vent 24 c can be directly connected to the exhaust space 41 .

[0044] When exhaust is performed through the exhaust space 41, the vent 24c, the ventilation path 24b, and the exhaust port 24a, similarly to the first embodiment, a negative pressure is applied to the reaction vessel 20 and further to the flow path 30, and the reaction liquid is accommodated in each reaction liquid accommodation space 21. Then, with the reaction liquid accommodated in the reaction liquid accommodation space 21, heating of the reaction liquid accommodation space 21 (or both the reaction liquid accommodation space 21 and the reaction liquid supply space 22) begins, and the reaction liquid is subjected to a nucleic acid amplification reaction.

[0045] The reaction vessel of the third aspect has both the characteristic of the reaction vessel of the first aspect, i.e., a volume ratio V2 / V1 of 3.0 or less, and the characteristic of the reaction vessel of the second aspect, i.e., a vent path extending from the exhaust port. Therefore, the reaction vessel of the third aspect exhibits both the effect of suppressing the occurrence of inspection abnormalities described in the first aspect and the effect of suppressing the occurrence of inspection abnormalities described in the second aspect, and is therefore able to further suppress the occurrence of inspection abnormalities.

[0046] <Nucleic Acid Amplification Cartridge> The present invention encompasses a nucleic acid amplification cartridge. The nucleic acid amplification cartridge of the present invention is a nucleic acid amplification cartridge for subjecting a reaction solution to a nucleic acid amplification reaction, and is a cartridge having at least one reaction chamber for containing the reaction solution and a flow path for supplying the reaction solution to the reaction chamber. The reaction chamber is the reaction chamber of the present invention described above. The flow path is connected to the reaction solution supply port of the reaction chamber. Components other than the reaction chamber and the flow path can include, for example, the components of a liquid-containing chamber and flow path switching means possessed by the cartridge described in Patent Document 4.

[0047] The reaction tank in the cartridge of the present invention may be any of the reaction tanks of the first, second, and third embodiments, and from the viewpoint of further suppressing the occurrence of inspection abnormalities, it is preferably a reaction tank of the third embodiment in which the volume ratio V2 / V1 is 3.0 or less and which has an air passage extending from the exhaust port downstream of the exhaust port.

[0048] <<When the reaction tank of the first embodiment is included>> When the reaction tank in the cartridge of the present invention is the reaction tank of the first embodiment, the exhaust port is directly connected to, for example, the exhaust space on the back side, thereby connecting the reaction tank to an external exhaust mechanism.

[0049] The cartridge of the present invention includes a cartridge body having recesses on its surface that define spaces such as the reaction liquid storage space 21, the reaction liquid supply space 22, and the flow path 30, and further having recesses on its back surface that define spaces such as the exhaust space 41, a front film attached to the front surface of the cartridge body, and a back film attached to the back surface of the cartridge body. The front film is attached to the front surface of the cartridge body, and functions as part of a side wall that seals the front space. The back film is attached to the back surface of the cartridge body, and functions as part of a side wall that seals the back space.

[0050] The cartridge body can be manufactured by, for example, resin molding. The material of the cartridge body is preferably a polypropylene-based resin because of its moldability, weldability, and disposability. The material of the front and back side films is also preferably a polypropylene-based resin from the same viewpoint.

[0051] The cartridge of the present invention can have one or more reaction vessels of the present invention. The number of reaction vessels of the cartridge of the present invention is preferably two or more, and can be 3, 4, 5, 6, 7, 8, 9, or 10 or more, but is not limited to these numbers. The cartridge of the present invention can also have reaction vessels other than the reaction vessel of the present invention, but it is preferable that all reaction vessels are reaction vessels of the present invention. Figures 1 and 2 show, as an example, a cartridge 10 having four reaction vessels of one embodiment of the present invention.

[0052] The flow path 30 is connected to the reaction solution supply port 23 of each reaction vessel 20 to transport the reaction solution to each reaction vessel. In the cartridge 10 of FIG. 1 , the flow path 30 has branch points 31a and 31b, at which one flow path branches into two and ultimately into four. The branches at branch points 31a and 31b are formed symmetrically, and the reaction solution is divided approximately equally at branch points 31a and 31b. Each of the branched flow paths is connected to the reaction solution supply port 23 of the reaction vessel 20. The reaction solution moving from upstream within the flow path is divided into two streams at branch point 31a, and each stream is further divided into two streams at branch point 31b, for a total of four streams. The divided reaction solution is each supplied to each reaction vessel 20 from the reaction solution supply port 23.

[0053] When the cartridge of the present invention has two or more reaction chambers, it is preferable that, for each reaction chamber, the length of the flow path from the branch point closest to the reaction chamber (the branch point located most downstream) to the reaction solution supply port is longer than the linear distance from the branch point to the reaction solution supply port (in other words, the flow path from the branch point closest to the reaction chamber to the reaction solution supply port is detouring). When the reaction solution is supplied from the reaction solution supply port 23, the reaction solution may bounce back and flow into the reaction solution supply port 23, resulting in the reaction solution remaining in the flow path near the reaction solution supply port 23. The remaining reaction solution may be forced by water vapor generated during the nucleic acid amplification reaction to flow backward through the flow path, pass through the branch point 31b closest to the reaction chamber, and be mixed into an adjacent reaction chamber. By detouring the flow path from the branch point closest to the reaction chamber to the reaction solution supply port, the length of the flow path can be secured, and the reaction solution remaining in the flow path near the reaction solution supply port can be prevented from flowing backward and reaching an adjacent reaction chamber. When the length of the flow path from the branch point closest to the reaction vessel to the reaction solution supply port is L and the linear distance from the branch point to the reaction solution supply port is Lo, the ratio of these lengths, L / Lo, is preferably more than 1.0 and not more than 3.0, and more preferably 1.2 to 2.0.

[0054] When the cartridge of the present invention has two or more reaction vessels, it is preferable that the position of the branch point closest to each reaction vessel is the same as or higher than the position of the exhaust part along the longitudinal direction of the reaction vessel. In the cartridge having the reaction vessel of the first embodiment, the exhaust part 24 is the exhaust port 24a, and the position of the exhaust part can be based on, for example, the center of the exhaust port 24a. By ensuring a longer distance between the branch point closest to the reaction vessel and the reaction solution supply port, it is possible to prevent the reaction solution remaining in the flow path near the reaction solution supply port from flowing back and reaching an adjacent reaction vessel.

[0055] When the cartridge of the present invention has two or more reaction vessels, the cartridge can further include an exhaust gas confluence space having a confluence point where all exhaust gas flows from each reaction vessel are combined into a single flow. In this case, the volumes of the spaces through which each exhaust gas passes from the exhaust port of each reaction vessel to the confluence point are set to be approximately equal. Setting the volumes of these spaces to be approximately equal has the advantage that negative pressure due to the exhaust gas is applied approximately evenly to each reaction vessel, resulting in approximately equal distribution of the reaction liquid to multiple reaction vessels. The confluence of the exhaust gas flows may be carried out in a single stage at a single confluence point, or in multiple stages at multiple confluence points. When there are multiple confluence points, the volumes to be compared are the volumes of the spaces through which each exhaust gas passes up to the final confluence point where a single flow is ultimately created.

[0056] 2, the cartridge 10 has, on its back surface, an exhaust space 41 including an exhaust merging space 42. The exhaust space 41 is in communication with each vent port 24c and a vent port 49, and the vent port 49 is in communication with an external exhaust mechanism directly or indirectly via another exhaust space. When exhaust is performed by the exhaust mechanism, a negative pressure is applied to each reaction vessel 20 via the vent port 49, the exhaust space 41, and each vent port 24c.

[0057] In the cartridge 10 shown in FIGS. 1 and 2 , a portion of the exhaust space 41 is an exhaust confluence space 42, which is formed by a partition 44 as a bilaterally symmetrical flow path space having confluence points 42a and 42b where exhaust streams converge. The exhaust confluence space 42 converges two exhaust streams from the reaction vessels 20 into one at confluence point 42a, and the converged streams converge again into one stream at confluence point 42b. Confluence point 42b is the final confluence point. In the exhaust confluence space 42, the volume of the space through which the exhaust gas passes from one vent 24c to confluence point 42b is approximately equal to the volume of the space through which the exhaust gas passes from the other vent 24c to confluence point 42b. Because the volumes of the spaces through which each exhaust gas passes from the exhaust port of each reaction vessel to the confluence point (or the final confluence point) are approximately equal to each other, negative pressure due to the exhaust gas is applied approximately evenly to each reaction vessel. As a result, the reaction liquid is distributed approximately evenly among the multiple reaction vessels, making it easier to ensure an appropriate amount of reaction liquid in each reaction vessel. In addition to being formed by a partition as described above, the exhaust gas confluence space 42 may also be formed by, for example, an air passage of a constant width and the same length that connects the vent 24c and the confluence point (or the final confluence point).

[0058] The cartridge of the present invention may include other components for preparing a reaction solution from a sample. These components include, for example, a chamber for storing the sample in the cartridge, a chamber for storing a test solution, a chamber for storing bead-like dry reagents, a chamber for storing wastewater generated during preparation of the reaction solution, an extraction section for extracting nucleic acid substances from the sample (e.g., using a glass filter), multiple flow paths connecting the chamber, the extraction section, and the reaction vessel, and a flow path switching section for appropriately switching between these flow paths. For example, a reaction solution can be prepared by transporting the sample stored in the cartridge to the extraction section, extracting substances from the sample in the extraction section, transporting a test solution to the extraction section, incorporating the extract into the test solution, and adding a dry reagent to the test solution. The prepared reaction solution is transported to flow path 30.

[0059] The cartridge 10 of the present invention can contain bead-shaped dry reagents containing primers or probes for nucleic acid amplification in the reaction solution containing spaces 21 or reaction solution supply spaces 22 of at least some of the reaction chambers 20. The dry reagents dissolve in the reaction solution when the reaction solution is supplied to the reaction chambers and are used in the nucleic acid amplification reaction. When the cartridge has multiple reaction chambers, the dry reagents may be the same or different for each reaction chamber.

[0060] <<In the Case of Having a Reaction Tank of the Second or Third Aspect>> When the reaction tank in the cartridge of the present invention is the reaction tank of the second or third aspect, the reaction tank 20 is connected to an external exhaust mechanism via the exhaust port 24a, the ventilation channel 24b, the ventilation port 24c, and further via the exhaust space 41 on the back side, as shown in Fig. 1. In this case, the other components are the same as in the case of the cartridge having the reaction tank of the first aspect, unless otherwise specified.

[0061] When the cartridge of the present invention has two or more reaction vessels, it is preferable that the position of the branch point closest to each reaction vessel is the same as or higher than the position of the exhaust unit along the longitudinal direction of the reaction vessel. In cartridges having reaction vessels of the second and third embodiments, the exhaust unit 24 includes an exhaust port 24a and an air vent 24b, and the position of the exhaust unit can be based on, for example, the midpoint of the air vent 24b in the longitudinal direction. For example, in the cartridge 10 shown in FIG. 1, for each reaction vessel 20, the position of the branch point 31b closest to the reaction vessel 20 is higher along the longitudinal direction of the reaction vessel 20 than the midpoint of the air vent 24b in the longitudinal direction. By ensuring a longer distance between the branch point closest to the reaction vessel and the reaction solution supply port, it is possible to prevent the reaction solution remaining in the flow path near the reaction solution supply port from flowing back into an adjacent reaction vessel.

[0062] When the cartridge of the present invention has two or more reaction tanks, the cartridge can further have an exhaust gas merging space having a junction where all exhaust gas flows from each reaction tank are merged into one flow. In this case, the volumes of the spaces through which each exhaust gas passes from the exhaust port of each reaction tank to the junction point are set to be approximately equal to each other. Detailed explanations are the same as for the cartridge having a reaction tank of the first embodiment. In addition, when the cartridge of the present invention has two or more reaction tanks, it is preferable that, for each reaction tank, the length of the flow path from the branch point closest to the reaction tank to the reaction liquid supply port is longer than the linear distance from the branch point to the reaction liquid supply port. Detailed explanations are the same as for the cartridge having a reaction tank of the first embodiment.

[0063] <Nucleic Acid Amplification Device> The present invention encompasses a device that subjects a reaction solution to a nucleic acid amplification reaction using the above-described cartridge of the present invention. The nucleic acid amplification device of the present invention is a nucleic acid amplification device that includes a mounting section for mounting the above-described cartridge of the present invention, an exhaust mechanism for evacuating the inside of the reaction chamber of the cartridge, and a heating mechanism for heating the reaction solution storage space and reaction solution supply space of the cartridge. The nucleic acid amplification device of the present invention is configured to be able to fully automatically prepare a reaction solution for a sample contained in the cartridge, transport the reaction solution to the reaction chamber, and subject the reaction solution to a nucleic acid amplification reaction.

[0064] In the nucleic acid amplification device of the present invention, when the cartridge is mounted in the mounting portion, the exhaust mechanism is connected to the vent 49 of the cartridge 10 , and the heating mechanism is disposed near the reaction chamber 20 .

[0065] As the exhaust mechanism, a general vacuum pump can be used. When the exhaust mechanism performs exhaust, negative pressure is applied to the reaction vessel 20 of the cartridge 10 through the vent 49.

[0066] The heating mechanism heats the reaction solution to subject it to a nucleic acid amplification reaction. The heating mechanism may be positioned opposite the reaction solution storage space and configured to heat the reaction solution storage space, or may be positioned opposite both the reaction solution storage space and the reaction solution supply space and configured to heat both the reaction solution storage space and the reaction solution supply space. When the heating mechanism heats the reaction solution storage space or the reaction solution supply space, the heat applied by the heating mechanism propagates to other areas of the cartridge via the gas within the cartridge or the cartridge body, resulting in an increase in the temperature of the entire cartridge despite a temperature gradient. To enable independent heating of the reaction solution storage space and the reaction solution supply space, it is preferable to provide a first heater for heating the reaction solution storage space and a second heater for heating the reaction solution supply space. The provision of the second heater enables independent control of the temperature of the reaction solution supply space. For example, by using the second heater to control the temperature of the reaction liquid supply space to be approximately the same as the temperature of the reaction liquid storage space, it becomes possible to suppress the condensation of water vapor on the inner wall surface itself.

[0067] The nucleic acid amplification device of the present invention is equipped with an excitation light source that emits excitation light to excite fluorescent labels that label reagents (primers, probes, etc.) used in nucleic acid amplification, and a detector that detects the fluorescence emitted by the fluorescent labels, and can automatically perform the process up to the detection of amplified nucleic acids.

[0068] <Nucleic Acid Amplification Method> The present invention encompasses a nucleic acid amplification method using the cartridge of the present invention, which includes the steps of: (1) creating a negative pressure in the reaction vessel by suction through the exhaust port, thereby supplying a reaction solution from the reaction solution supply port to the reaction solution supply space, and (2) subjecting the reaction solution that has spontaneously moved from the reaction solution supply space to the reaction solution storage space to a nucleic acid amplification reaction.

[0069] In step (1), the reaction solution is supplied to the reaction solution supply space from the reaction solution supply port. The reaction solution is supplied by creating a negative pressure in the reaction tank by suction through the exhaust port. The reaction solution is supplied to the reaction solution supply space from the reaction solution supply port 23, which is located below the exhaust port 24a and above the protrusion 25, thereby preventing bubbles from forming in the reaction solution and further reducing the occurrence of test abnormalities in detection after nucleic acid amplification. Furthermore, it is preferable that the amount of reaction solution supplied to the reaction solution supply space be a volume equivalent to the volume of the reaction solution storage space. This makes it easier for the reaction solution to be stored in one mass in the reaction solution storage space due to the action of the protrusion, further reducing the occurrence of test abnormalities in detection after nucleic acid amplification.

[0070] In step (2), the reaction solution that spontaneously migrates from the reaction solution supply space to the reaction solution storage space is subjected to a nucleic acid amplification reaction. The nucleic acid amplification reaction can be controlled by heating, and the heating temperature is selected depending on the type of nucleic acid amplification enzyme, but is generally in the range of 50 to 70°C. The reaction solution is a sample solution that contains or may contain the nucleic acid to be amplified, and also contains a nucleic acid amplification enzyme and a nucleic acid amplification primer or probe. The enzyme and primer or probe can be added to the reaction solution before it is supplied to the reaction vessel, or they can be added in the reaction vessel. Addition in the reaction vessel can be achieved by placing water-soluble beads containing a nucleic acid amplification enzyme and water-soluble beads containing a nucleic acid amplification primer or probe between the reaction solution supply port and the protrusion of the reaction solution supply space.

[0071] The nucleic acid to be amplified in the reaction solution is not particularly limited and can be appropriately selected depending on the intended use of the cartridge of the present invention. Furthermore, the nucleic acid amplification enzyme, nucleic acid amplification primer, and probe are not particularly limited and appropriate materials can be used depending on the nucleic acid amplification method.

[0072] The nucleic acid to be amplified is not particularly limited, and may be, for example, RNA or DNA. The nucleic acid amplification reaction may be an isothermal amplification reaction or a thermocycling amplification reaction of nucleic acid. The isothermal amplification reaction may be, for example, the LAMP method or the SmartAmp method. The thermocycling amplification reaction may be a PCR amplification reaction.

[0073] The nucleic acid amplification enzyme is not particularly limited, and may be, for example, an enzyme for isothermal nucleic acid amplification reaction or an enzyme for thermocycling amplification reaction. The isothermal nucleic acid amplification reaction may be, for example, the LAMP method or the SmartAmp method, and may be an enzyme for nucleic acid amplification reaction using a strand displacement reaction.

[0074] The polymerase, which is an enzyme for nucleic acid amplification reactions having strand displacement activity, can be any known enzyme. Examples include, but are not limited to, the polymerases described in International Publication No. 2004 / 040019. The polymerase having strand displacement activity can also be DNA polymerase (Aac), which is disclosed in International Publication No. 2009 / 054510 (Japanese Patent No. 4,450,867).

[0075] As a polymerase having strand displacement activity for use in a nucleic acid amplification reaction, any of mesophilic, mesophilic, and thermostable polymerases can be suitably used. Furthermore, this polymerase may be either a natural form or an artificially mutated mutant. Examples of such polymerases include DNA polymerases. Examples of such DNA polymerases include mutants of DNA polymerases derived from thermophilic Bacillus bacteria, such as Bacillus stearothermophilus (hereinafter referred to as "B.st") and Bacillus caldotenax (hereinafter referred to as "B.ca"), lacking 5' to 3' exonuclease activity, and the Klenow fragment of DNA polymerase I derived from Escherichia coli (E. coli). Further examples of DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9°Nm DNA polymerase, Therminator DNA polymerase, and Taq DNA polymerase.

[0076] When the nucleic acid to be amplified is RNA, a reverse transcriptase can be used in combination with a DNA polymerase, or a DNA polymerase that also has reverse transcription activity can be used as the DNA polymerase. The reverse transcriptase is not particularly limited as long as it has cDNA synthesis activity using RNA as a template, and examples include reverse transcriptases from various sources, such as avian myeloblastosis virus reverse transcriptase (AMV-RTase), Rous-associated virus 2 reverse transcriptase (RAV-2RTase), and Moloney murine leukemia virus reverse transcriptase (MMLV RTase). Examples of DNA polymerases that also have reverse transcription activity include BcaBEST DNA polymerase, Bca(exo-)DNA polymerase, and Tth DNA polymerase.

[0077] The primers are appropriately selected depending on the enzyme used for the nucleic acid amplification reaction. When the enzyme for the nucleic acid amplification reaction is an enzyme for nucleic acid amplification reaction utilizing a strand displacement reaction, examples of the primers include those described in WO 2004 / 040019, JP 2009-171935 A, JP 2011-50380 A, and the like.

[0078] The method of the present invention can further include a step of optically detecting, electrically detecting, or detecting by surface plasmon resonance the nucleic acid amplified in the reaction vessel after the nucleic acid amplification reaction. The amplified nucleic acid can be detected using a fluorogenic primer as the primer and using the label of the fluorogenic primer. The amplified nucleic acid can be detected by utilizing the exciton effect using an exciton primer or exciton probe in the amplification reaction. The method for optically detecting nucleic acid may be a method using an intercalating dye.

[0079] In the method of the present invention, it is preferable that the nucleic acid amplification reaction is carried out by the SmartAmp method or the LAMP method, and the label is detected using an exciton primer or an exciton probe. Alternatively, it is preferable that the nucleic acid amplification reaction is carried out by the PCR method, and the label is detected using an exciton primer or an exciton probe.

[0080] Following the nucleic acid amplification reaction, a nucleic acid melting curve can be plotted, and the melting curve can be used to determine the properties of the amplified product, such as whether it is a false positive or a true positive.

[0081] The present invention will be described in more detail below with reference to examples. The materials, amounts used, proportions, treatment contents and treatment procedures shown in the examples can be appropriately changed within the scope of solving the problems of the present invention.

[0082] Nucleic acid amplification reaction was carried out by the SmartAmp method using the following materials.

[0083] <Sample> Sample RNA: 1 x 10 ３ Sample RNA Negative control: water (H ２O) <Enzyme> DNA polymerase (Aac) 34 U / reaction Reverse transcriptase (AMV-RT) 2 U / reaction <Primer set> Folding primer (FP) Outer boost primer (oBP) Turn-back primer (TP), turn-back site Internal boost primer (iBP) Turn-back primer (TP), annealing site Outer primer (OP) <Internal positive control (IPC)> 2 x 10 ５ Rubisco RNA <Amplification reaction buffer> Trizma pH 8.0 20mM, CH ３ COOK 60mM, (NH ４ ) ２ SO ４ 20 mM MgSO ４ 16mM, Tween 20 0.2% (w / v), dNTP 1.4mM, RNase free-water

[0084] The volume of reaction solution distributed to each reaction chamber was set to 20 μL. Nucleic acid amplification reactions 1 to 4 shown below are examples (Examples) of tests using a cartridge with a reaction chamber of the present invention as shown in Figure 1. In the reaction chamber of the Example, the volume ratio is 1.5 (30 μL / 20 μL), the longitudinal length of the reaction chamber is 7 mm, and the length, width, and height of the air vent are 4 mm, 0.7 mm, and 0.4 mm, respectively. In addition, a portion of the upper surface of the inner wall of the reaction chamber has a 125-degree inclination angle. Nucleic acid amplification reactions 5 to 7 are examples (Comparative Examples) of tests using the conventional cartridge shown in Figure 5, in which an abnormality occurred. In the reaction chamber of the Comparative Example, the volume ratio is 5.4 (108 μL / 20 μL), and the longitudinal length of the reaction chamber is 13 mm. In the Comparative Example, there is no air vent, and the exhaust port is directly connected to the exhaust space on the back side.

[0085] <Nucleic Acid Amplification Reaction 1> In the arrangement shown in Figure 1, the first, second, third, and fourth reaction chambers are shown from the left. Water-soluble beads containing a primer set for IPC were placed in the first reaction chamber, and water-soluble beads containing a primer set for sample RNA were placed in the second reaction chamber. A film was then attached to the surface of the cartridge body to create a nucleic acid amplification cartridge. No primer set was placed in the third and fourth reaction chambers. Using the cartridge, a reaction solution containing sample RNA, enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chambers through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0086] <Nucleic Acid Amplification Reaction 2> A nucleic acid amplification cartridge was prepared by placing water-soluble beads containing a primer set for IPC in the first reaction chamber, placing water-soluble beads containing a primer set for sample RNA in the fourth reaction chamber, and attaching a film to the surface of the cartridge body. No primer set was placed in the second or third reaction chamber. Using the cartridge, a reaction solution containing sample RNA, enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chamber through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0087] <Nucleic Acid Amplification Reaction 3> Water-soluble beads containing a primer set for IPC were placed in the first reaction chamber, and water-soluble beads containing a primer set for sample RNA were placed in the second to fourth reaction chambers. A film was attached to the surface of the cartridge body to prepare a nucleic acid amplification cartridge. Using the cartridge, a reaction solution containing sample RNA, enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chamber through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0088] <Nucleic Acid Amplification Reaction 4> Water-soluble beads containing a primer set for IPC were placed in the second reaction chamber, and a film was attached to the surface of the cartridge body to prepare a nucleic acid amplification cartridge. No primer set was placed in the first, third, or fourth reaction chambers. Using the cartridge, a reaction solution containing water (negative control), enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chambers through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0089] <Nucleic Acid Amplification Reaction 5> In the arrangement of Figure 5, the first, second, third, and fourth reaction chambers are shown from left to right. Water-soluble beads containing a primer set for IPC were placed in the second reaction chamber, and water-soluble beads containing a primer set for sample RNA were placed in the third reaction chamber. A film was then attached to the surface of the cartridge body to create a nucleic acid amplification cartridge. No primer sets were placed in the first and fourth reaction chambers. Using the cartridge, a reaction solution containing sample RNA, enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chambers through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0090] <Nucleic Acid Amplification Reaction 6> A nucleic acid amplification cartridge was prepared by placing water-soluble beads containing a primer set for IPC in the first reaction chamber and attaching a film to the surface of the cartridge body. No primer set was placed in the second to fourth reaction chambers. Using the cartridge, a reaction solution containing water (negative control), enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chamber through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0091] <Nucleic Acid Amplification Reaction 7> A nucleic acid amplification cartridge was prepared by placing water-soluble beads containing a primer set for IPC in the first reaction chamber and attaching a film to the surface of the cartridge body. No primer set was placed in the second to fourth reaction chambers. Using the cartridge, a reaction solution containing sample RNA, enzyme, IPC, and buffer was prepared, and the reaction solution was supplied to the reaction chamber through the reaction solution supply port. The reaction solution contained in the reaction solution storage space was then subjected to a nucleic acid amplification reaction.

[0092] <Explanation of Results> Figures 3AB and 3CD (a) to (d) show the results of nucleic acid amplification reactions 1 to 4 (Examples), respectively, and Figure 4 (a) to (c) show the results of nucleic acid amplification reactions 5 to 7 (Comparative Examples), respectively. "N / A" in the figures indicates the detection results in a reaction vessel in which the reaction solution did not contain a primer (fluorescent label).

[0093] In nucleic acid amplification reactions 1 to 4 (Examples), no false negative or false positive test abnormalities were observed, and normal results were obtained. Nucleic acid amplification reaction 5 is an example in which a false negative result was obtained due to insufficient amplification of sample RNA, and nucleic acid amplification reaction 6 is an example in which a false positive result was obtained in the negative control. In addition, nucleic acid amplification reaction 7 is an example in which slight nucleic acid amplification was observed in a reaction vessel without primers. The nucleic acid amplification in nucleic acid amplification reaction 7 is thought to be due to a portion of the reaction solution in the first reaction vessel being mixed with the reaction solution in the second reaction vessel. Furthermore, in tests using a cartridge having the reaction vessel of the present invention shown in Figure 1, the incidence of test abnormalities was significantly reduced. These results demonstrate that the use of a cartridge having the reaction vessel of the present invention can suppress the occurrence of test abnormalities.

[0094] The present invention is useful in fields related to nucleic acid amplification, nucleic acid detection, and virus testing.

[0095] REFERENCE SIGNS LIST 10 Cartridge 20 Reaction vessel 21 Reaction liquid storage space 22 Reaction liquid supply space 23 Reaction liquid supply port 24 Exhaust section 24a Exhaust port 24b Ventilation path 25 Projection 26 Opening 30 Flow path 31a, 31b Flow path branch point 41 Exhaust space 42 Exhaust merging space 42a, 42b Exhaust merging point 44 Partition

Claims

1. A reaction vessel having a sealed reaction vessel for nucleic acid amplification, the reaction vessel being provided with a reaction liquid supply port and an exhaust section, the exhaust section having an exhaust port, the lower space of the reaction vessel being a space for containing reaction liquid, the upper space being a space for supplying reaction liquid, the reaction liquid supply port and the exhaust port being located on the inner wall surface of the space for supplying reaction liquid, and the reaction liquid supply port being located below the exhaust port, and the ratio of the volume (V2) of the space for supplying reaction liquid to the volume (V1) of the space for containing reaction liquid (volume ratio V2 / V1) being 3.0 or less.

2. The reaction vessel according to claim 1, wherein the volume ratio V2 / V1 is 2.5 or less.

3. The reaction vessel according to claim 1 or 2, wherein at least a portion of the upper surface of the inner wall of the reaction vessel is inclined.

4. The reaction vessel according to claim 1 or 2, wherein at least the inner wall surface of the reaction liquid supply space is treated to be water repellent.

5. The reaction vessel according to claim 1 or 2, wherein the exhaust section further comprises an air passage extending from the exhaust outlet downstream of the exhaust outlet.

6. A sealed reaction vessel for nucleic acid amplification having a reaction liquid supply port and an exhaust section, wherein the exhaust section has an exhaust port and an air passage extending from the exhaust port downstream of the exhaust port, the lower space of the reaction vessel is a space for containing reaction liquid, the upper space is a space for supplying reaction liquid, the reaction liquid supply port and the exhaust port are located on the inner wall surface of the space for supplying reaction liquid, and the reaction liquid supply port is located below the exhaust port.

7. The reactor of claim 6, wherein the ratio of length to width of the vent passage is from 1 to 40.

8. The reaction vessel according to claim 6 or 7, wherein the length of the vent passage is 2 to 8 mm.

9. The reaction vessel according to claim 6 or 7, wherein the width of the vent passage is 0.2 to 2.0 mm.

10. The reaction vessel according to claim 6 or 7, wherein the inner wall surface of the ventilation passage is treated to be water repellent.

11. The reaction vessel of claim 1 or 6, wherein the exhaust port is located above the inner wall of the reaction vessel.

12. A nucleic acid amplification cartridge for subjecting a reaction liquid to a nucleic acid amplification reaction, comprising at least one reaction vessel for containing the reaction liquid, and a flow path for supplying the reaction liquid to the reaction vessel, the reaction vessel being the reaction vessel described in claim 1 or 6, and the flow path being connected to the reaction liquid supply port of the reaction vessel.

13. The cartridge according to claim 12, wherein the number of reaction vessels is two or more, the flow path is divided at a branching point and connected to the reaction liquid supply port of each reaction vessel, the cartridge further has an exhaust junction space having a junction where all exhaust flows from each reaction vessel are joined into one flow, and the volumes of the spaces through which each exhaust flows pass from the exhaust ports of each reaction vessel to the junction point are approximately equal to each other.

14. The cartridge described in claim 12, wherein the number of reaction vessels is two or more, the flow path is divided at a branch point and connected to a reaction liquid supply port of each reaction vessel, and for each reaction vessel, the length of the flow path from the branch point closest to the reaction vessel to the reaction liquid supply port is longer than the straight-line distance from the branch point to the reaction liquid supply port.

15. The cartridge according to claim 12, wherein the number of reaction vessels is two or more, the flow path is divided at a branch point and connected to a reaction liquid supply port of each reaction vessel, and the position of the branch point closest to each reaction vessel is the same as the position of the exhaust part or higher than the position of the exhaust part.

16. A nucleic acid amplification device comprising: a mounting section for mounting the cartridge according to claim 12; an exhaust mechanism for exhausting the inside of a reaction chamber of the cartridge; and a heating mechanism for heating a reaction liquid storage space and a reaction liquid supply space of the cartridge.

17. A method for nucleic acid amplification processing using the cartridge described in claim 12, comprising: supplying a reaction liquid from a reaction liquid supply port to a reaction liquid supply space by creating a negative pressure inside the reaction tank through suction from an exhaust port; and subjecting the reaction liquid which spontaneously moves from the reaction liquid supply space to the reaction liquid storage space to a nucleic acid amplification reaction.

Images